Fig. 2

Enhanced gene expression using the untranslated sequences from the CMV IE gene. The eukaryotic expression plasmid pCMVintCAT was obtained from Vical Inc. (San Diego, CA) and expresses the chloramphenicol acetyl transferase gene under the control of the CMV IE enhancer/promoter. This plasmid also contains the 5′ untranslated and first intron from the CMV IE gene and a bovine growth hormone polyadenylation sequence. The plasmid pCMVCAT contains the same backbone as pCMVintCAT with the entire 5′ untranslated and first intron sequences deleted (BsmBI to EcoRV). Porcine aortic vascular smooth muscle cells were obtained by standard explantation methods and used for transfection studies within the first six passages. Cells were grown to 30–40% confluence in 6-well plates in Medium 199 (Gibco/BRL) containing 10% fetal bovine serum and transfected with 5 µg of plasmid DNA and 10 µg of a cationic liposome, DMRIE/DOPE (Vical) (23). Transfections were performed in triplicate. Detection of CAT activity was performed as previously described (14). *p = 0.002 using unpaired two-tailed Student’s t-est.